Microarray methods for studying DNA and RNA have revolutionized biology, but progress towards analogous, highly parallel arrays for studying proteins has been slower. I propose combining existing high throughput sequencing, solid-phase DNA amplification and cell-free expression technologies to create a new type of high density protein array in which clonal protein features are generated in situ from immobilized DNA templates. Such a randomly addressed protein array would have the feature density necessary to enable parallel measurement of the properties of millions of proteins via activity, binding and substrate dependence assays. Protein identity and expression levels would be elucidated with existing high throughput sequencing methods. These protein arrays could be used to study protein properties and expression levels in a wide variety of previously difficult to access systems, including bacterial communities. Such communities are important in human health;for example, obese individuals harbor a set of gut microflora that is distinct from that of lean individuals and capable of harvesting more food energy. Obesity affects a third of Americans and is a risk factor for diabetes, heart disease, hypertension, and some types of cancers. Differences in gut microflora composition likely contribute to obesity, but disentangling the complex metabolic processes occurring in the gut is difficult. The gut contains 500-1000 different species estimated to collectively harbor between 2 and 4 million genes - a number that far exceeds the capacity of traditional biochemical analysis. To meet this challenge, I will use randomly addressed protein arrays to compare the protein activity and expression profiles of lean versus obese gut microflora. Therefore, I propose the following specific aims: 1) generation of randomly addressed DNA features using bridge PCR;2) cell-free transcription and translation of immobilized template DNA;and 3) functional analysis of the obesity associated protein complement. Usage of randomly addressed protein arrays should facilitate a better understanding of obesity and this technology could eventually become a widely used tool for high throughput analysis of protein function. PUBLIC HEALTH RELEVANCE This research seeks to understand obesity as it relates to the bacteria inside the gut that help digest the food we eat. This proposal describes a novel technology that should discover why certain types of gut bacteria are associated with obesity. A better understanding of this potential cause of obesity could lead to an effective treatment.